The creation and elimination of uracils in DNA plays a central role in the affinity maturation of B lymphocytes. Activation-induced deaminase (AID) converts cytosines in single-stranded DNA to uracil and the processing of these uracils by DNA repair pathways causes base substitution mutations (somatic hypermutation; SHM) and strand breaks that lead to region-specific recombination (class-switch recombination; CSR) in immunoglobulin (Ig) genes. AID is essential for both SHM and CSR. Most studies of SHM and CSR use mutations, strand breaks or translocations as proxies for the uracils created by AID. This is problematic because uracils in DNA may be repaired through error-prone or error-free pathways leading to many possible outcomes including replacement of uracils with cytosines, transition and transversion mutations, and strand breaks. In particular, a restoration of cytosines at these uracils erases the evidence of AID-mediated cytosine deamination. All current technologies for detecting, quantifying and mapping uracils are indirect. We propose here development of the first technology to directly map uracils created by the AID/APOBEC family deaminases which contribute to both innate and acquired immunity in humans at the genomic level. Furthermore, we propose to use it to directly visualize the uracils at a single cell level. A mycobacterial protein, UdgX, links covalently to uracils in DNA with a strong preference for UG pairs. It forms denaturation-resistant complexes with uracils and will be used to pull-down and sequence genomic DNA fragments containing uracils. To facilitate this, the protein has been appended with FLAG and other tags, and these tags allow for affinity pull-down of DNA-protein complexes. To demonstrate the feasibility of this approach, we will pull down DNA fragments from uracil excision-deficient Escherichia coli expressing APOBEC3A (A3A), prepare and sequence libraries derived from them, and map them to the E. coli genome. To determine the accuracy of the new uracil pull-down technique, genomic map of uracils created by A3A using UdgX will be compared with a recently created map of uracils created by A3A using an alternate pull-down technique. In a second approach to demonstrate usefulness of this technology, UNG?/? CH12F3 cells will be stimulated to undergo CSR and transfected with mCherry-tagged UdgX. The cells will be fixed and the Ig switch regions and ? will be visualized using FISH probes. A colocalization of the mCherry signal with the FISH signal will confirm that UdgX has bound at the IgH locus. We will also use the FLAG tag to pull-down the CH12F3 genomic fragments linked to UdgX, and use qPCR and deep sequencing to confirm that UdgX preferentially pulls down S and S? regions. Our long-term goal is to combine the technologies created under this proposal to map uracils created by AID during antibody maturation to V(D)J and appropriate switch regions in the Ig genes, and to off- target sites across the whole genome of B cells undergoing germinal center maturation, and study the kinetics of uracil elimination by the repair processes.